Steps for Standard PCR Reaction
Design primers. In general, primers should have the following properties:
Length of 18-24 bases
40-60% G/C content
Start and end with 1-2 G/C pairs
Melting temperature (Tm) of 50-60o C
Primer pairs should have a Tm within 5o C of each other
Primer pairs should not have complementary regions
Tip: Primer3 is an excellent resource for choosing primers.
Tip: If you will be including a restriction site at the 5'' end of your primer, note that a 3-6 base pair spacer should be added in order for the enzyme to cleave efficiently.
Set up PCR tubes.
Place thin-walled PCR tubes on ice.
For a 50 μL reaction, add:
2 μL Template DNA (10 ng-500 ng)
5 μl 10X Taq buffer with MgCl2
1 μl dNTP mix (10 mM each nt)
2.5 μL Forward Primer (10 μM stock)
2.5 μL Reverse Primer (10 μM stock)
0.2 μL Taq DNA Polymerase (5 units/μL)
32.8 μL Sterile deionized water (variable)
Tip: If you are doing multiple PCR reactions, save time by creating a "master mix."
PCR: The following is a typical PCR program. The annealing temperature should be 5o C below the primer Tm. The extension step should be 1-2 minutes per kilobase of product, depending on whether you are using a polymerase with proofreading capabilities. See manufacturer''s instructions.
Step 1: Initial Denaturation for 2 minutes at 95o C
Step 2: Denature for 1 minute at 95o C
Step 3: Anneal primers for 30 seconds at 55o C (or 5o C below Tm)
Step 4: Extend DNA for 2 minutes at 72o C
Step 5: Repeat steps 2-4 for 25-30 cycles
Step 6: Final Extension for 10 minutes at 72o C
Run 2 μL on a gel to check size and concentration of PCR product.